| Backgroud and ObjectiveThe colon cancer is one of the most common malignant tumors of digestive system, and in recent years the incidence of which has been increasing in worldwide. Researches have shown that environmental and chemical factors are chiefly responsible for the rise of the incidence. One of the main carcinogens for colon cancer is heterocyclic amines which exist in meat with high-temperature cooking processed. These carcinogens, after being activated, can bind to DNA of the colonic epithelial cells and form an DNA adduct, resulting in carcinogenesis of the normal colonic tissues. Therefore, looking for safe and effective ways to enhance the metabolism and detoxification of carcinogen and reduce the activation of carcinogen, has important significance in public health with the respect to diminish the incidence rate of colon cancer Sulforaphane (SFN), an important phytochemicals, is highly enriched in cruciferous plants. Researches on epidemiology and pharmacology have shown that SFN possesses important role of chemopreventive effects on cancers of lung, liver, stomach, colon, prostate and breast. In the stage of cancer initiation, SFN can prevent the occurrence of cancer mainly through the following two mechanisms. On one hand, through the activation of Kelch-like ECH-associated proteinl (Keapl)-NF-E2-related factor2(Nrf2)-antioxidant response element (ARE) pathway, SFN can enhance its ability of detoxification and removal of carcinogen by inducing Phase2enzymes, including glucuronosyltransferase (UGT). On the other hand, SFN can also block carcinogen activation via inhibition of cytochrome P450(CYP), an representative of Phase1enzymes, such as CYP1A1, CYP2B1/2, CYP3A4and et al.Autophagy is a hot research point in recent years. It plays an important role in the genesis and development of cancer. Recent studies revealed that SFN at relative low concentrations could induce autophagy in human prostate, colon and breast cancer cells. These studies also indicated that addition of autophagy inhibitor such as3-MA or bafilomycin Al can potentiate SFN-induced cell cycle arrest and apoptosis which suggest the use of an autophagy inhibitor for enhancement of the chemopreventive effects of SFN. Rapamycin (Rapa), as a commonly used autophagy activator, could up-regulate autophagy via inhibition on mammalian target of Rapa (mTOR). Rapa could inhibit various cancer cells growth, and has a synergistic antitumor effect when combined with other antineoplastic drugs.The studies of SFN mentioned above mainly care about carcinogenesis in advanced stage, and attempted to potentiate the effects of cell growth arrest or cell death through co-treatment with SFN and autophagy modulators. However, it plays an important role in cancer prevention that SFN can diminish the aggression of carcinogens via induction of Phase2enzymes and inhibition of Phase1enzymes at the stage of cancer initiation. In the present study, experiments were performed in the human colon cancer Caco-2cells, an excellent model for studying UGT and CYP3A4expression, which have characteristics of normal intestinal epithelial cells. Based on this model, we assayed the expression of CYP3A4, UGT1A1, UGT1A8and UGT1A10, as representative members of Phase1and Phase2enzymes, and explored their mechanisms in cells co-treated with SFN and either3-MA or Rapa. Our studies may provide new insights for chemoprevention of colon cancer.Methods1. The Caco-2cells were divided into six experimental groups:the control (no drug treatment), SFN,3-MA, Rapa, SFN plus3-MA and SFN plus Rapa.2. The proliferative status and morphological change of Caco-2cells were visualized by an inverted phase contrast microscope. The inhibition rate of cell viability was assessed using the cell counting kit-8(CCK-8) assay. We select the appropriate concentration and duration of drug treatment based on results of the microscopic observation and CCK-8assay.3. Western blot was employed to detect the expression of microtubule-associated protein1light chain3(LC3). Immunocytochemistry was performed to observe the intracellular distribution of LC3. Transmission electron microscope was used to observe ultrastructures of Caco-2cell.4. Quantitative real-time RT-PCR was taken to examine the mRNA expression of UGT1A1, UGT1A8, UGT1A10, hPXR and CYP3A4. Western blot was employed to detect the expression of UGT1A1. Immunocytochemistry was performed to observe the nuclear localization of Nrf2.Results1. Based on results of the microscopic observation and CCK-8assay, concentrations of25μM SFN,2.5μM3-MA and10nM Rapa were chosen in subsequent experiments.2. A dose-and time-dependent manner increase in LC3-Ⅱ levels was observed in Caco-2cells treated with SFN, and3-MA reduced LC3-Ⅱ protein levels while Rapa enhanced its expression. Autophagysomes and autolysosomes can be visualized in the cells treated with SFN, Rapa and their combination by transmission electron microscope.3. In comparison to the control group, UGT1A1, UGT1A8, UGT1A10mRNA levels were increased significantly after treatment of SFN and SFN/Rapa combination, and SFN/Rapa combination possessed the highest level (all P-values less than0.05). The UGT1A isoforms expression of SFN/3-MA combination had no statistic difference compared with the control group (all P-values more than0.05). Treatment with SFN alone and SFN/3-MA combination caused some Nrf2accumulation in the nuclear region, and SFN/Rapa combination treatment caused even more Nrf2nuclear staining. The Rapa alone and SFN/Rapa combination treatment groups had higher levels of hPXR mRNA compared with the control group (all P-values less than0.05).4. Compared with the control group, SFN-treated, Rapa-treated, and SFN/Rapa-treated cells had reduced levels of CYP3A4mRNA. Compared with the SFN treatment group, SFN/Rapa combination had no statistic difference in CYP3A4mRNA expression, and SFN/3-MA combination, however, increased the level of CYP3A4which had significant difference (all P-values less than0.05).Conclusions1. Co-treatment with Rapa could enhance SFN-induced autophagy, and increase mRNA expression of UGT1A1, UGT1A8and UGT1A10induced by SFN. The mechanism involved in the synergistic induction of UGT1A isoforms may be the combined activation of Nrf2and hPXR signaling pathways. Further, the up-regulation of hPXR did not cause CYP3A4induction. Our data offer evidence to suggest the potential use of an autophagy activator for enhancement of the chemopreventive effects of SFN on colon cancer, particularly through the induction of Phase2enzymes.2.3-MA may suppress SFN-induced autophagy and UGT1A isoforms mRNA expressions. In addition,3-MA can enhance CYP3A4mRNA expression which is suppressed by SFN. These results indicate that, at the stage of initiative mutation, autophagy inhibitor may exert negative impacts on chemopreventive effects of SFN on colon cancer by interfering induction of Phase1enzymes and suppression of Phase2enzymes. |
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